Overexpression of miR390b promotes stem elongation and height growth in Populus

Abstract MicroRNA390 (miR390) is involved in plant growth and development by down-regulating the expression of the downstream genes trans-acting short interfering RNA3 (TAS3) and AUXIN RESPONSE FACTORs (ARFs). There is a scarcity of research on the involvement of the miR390-TAS3-ARFs pathway in the stem development of Populus. Here, differentially expressed miRNAs during poplar stem development were screened by small RNA sequencing analysis, and a novel function of miR390b in stem development was revealed. Overexpression of miR390b (OE-miR390b) resulted in a large increase in the number of xylem fiber cells and a slight decrease in the cell length at the longitudinal axis. Overall increases in stem elongation and plant height were observed in the OE-miR390b plants. According to transcriptome sequencing results and transient co-expression analysis, TAS3.1 and TAS3.2 were identified as the target genes of miR390 in poplar and were negatively regulated by miR390 in the apex. The transcription levels of ARF3.2 and ARF4 were significantly repressed in OE-miR390b plants and strongly negatively correlated with the number of xylem fiber cells along the longitudinal axis. These findings indicate that the conserved miR390-TAS3-ARFs pathway in poplar is involved in stem elongation and plant height growth.


Introduction
Populus is a model plant for studying the growth and development of trees and perennial woody plants. Meanwhile, Populus is a significant afforestation species and a major raw material for various timber industries. Secondary growth of woody plants is the basis for the wood formation and its prerequisites are primary growth and stem elongation [1]. Elongation growth usually occurs during the transition from primary to secondary growth of stem, and the associated molecular regulatory mechanisms are critical for improving wood quality and yield through genetic engineering. Some studies have been conducted to validate the regulatory functions of genes related to stem elongation and plant height in poplar. For example, overexpression of the heterologous PHY-TOCHROME A (PHYA) was found to result in the accumulation of CENTRORADIALIS-LIKE1 (CENL1) in the rib meristem under shortday treatment, thereby promoting stem elongation and affecting plant height [2]. Overexpression of the cytokinin metabolism gene CYTOKININ OXIDASE (CKX) led to a decrease in cytokinin levels, which further significantly inhibited stem elongation [3]. Expression of both GA20-oxidase, a key gene of the gibberellin synthesis pathway, and GID1, a GA receptor gene, positively regulated internode elongation and plant height growth [4,5]. Recently, two tandem CCCH zinc finger genes, C3H17 and C3H18, targeted by MYB3 and MYB21, were reported to affect stem elongation and plant height [6,7]. However, the molecular mechanisms regulating stem elongation and plant height in poplar remain unclear.
As key regulatory factors in plant growth and development, microRNAs (miRNAs) are a class of small single-stranded noncoding RNAs (∼21 nucleotides) that are involved in the negative regulation of target gene expression primarily by inhibiting the initiation of mRNA translation of the target gene or by directly cleaving the mRNA of the target gene [8]. Numerous miRNAs were demonstrated to serve as important regulators of wood formation. Specifically, the functional inhibition of both miR393 and miR6443 increased the lignin content, and the overexpression of both miR397 and miR6443 decreased the lignin content, indicating that miR393, miR397, and miR6443 are negative regulators of lignin biosynthesis in poplar [9][10][11]. The gene TEOSINTE BRANCHED 1/CYCLOIDEA/PCF 20 (TCP20), which is negatively regulated by miR319, promotes vascular formation layer proliferation through interaction with WUSCHEL-RELATED HOMEOBOX 4 (WOX4) and activates WND6 transcription to promote secondary xylem differentiation [12]. By regulating the target class III homeodomainleucine zipper (HD-Zip III), miR166 participates in poplar secondary growth and the initiation of cambium [13,14]. Moreover, various omics data have been continuously reported in terms of stem development in different poplar varieties [15][16][17][18]. However, there is a scarcity of research on miRNA in poplar stem elongation.
In plants, miR390 cleaves the transcript of the target gene trans-acting siRNA 3 (TAS3) to produce trans-acting small interfering RNAs (tasiRNAs), also known as tasiARFs, which can cleave transcripts of AUXIN RESPONSE FACTOR (ARF) 2, ARF3, and ARF4 [19,20]. The regulatory functions of miR390/TAS3/ARFs have been demonstrated in several species, such as the involvement in lateral root (LR) formation, f loral meristem determinacy, and seed size in Arabidopsis [21][22][23][24], somatic embryogenesis of longan [25], the establishment of leaf polarity in maize [26], development phase transition of Physcomitrella patens [27], root nodule symbiosis of Lotus japonicus [28], nodulation formation of Medicago truncatula [29], maintenance of normal development of vascular tissue and shoot apical meristem (SAM) in rice [30], and response to biotic stress [31] and abiotic stress [32]. In Populus, the expression level of miR390 was positively correlated with vascular cambial activity [16]. The functions of miR390 in response to salt stress promoting LR development has been recently verified in Populus [33]. However, there is limited research on the involvement of miR390/TAS3/ARFs in plant stem elongation. In the present study, differentially expressed miR390 was mined by small RNA sequencing (sRNA-Seq) of different poplar tissues during stem development, and its function was verified by the transgene verification. Our findings showed that miR390 has a novel function in poplar stem elongation and plant height growth.

miR390b is ubiquitous in poplar
The miR390 family has 374 members from 163 plant species [34], of which 306 members share the same mature sequence (5'-AAGCUCAGGAGGGAUAGCGCC-3 , as the standard mature sequence of miR390), as shown in sequence logo ( Fig. 2A). Among the sequences, the mature sequences of miR390a-c/d-5p in poplar were consistent with the standard sequence. RT-qPCR of the primary transcripts revealed four MIR390 genes with distinct expression patterns in P. deltoides. Pri-miR390a/b/d were highly expressed in AP and IN9, while pri-miR390c was specifically expressed in IN9 (Fig. S2, see online supplementary material).
In this study, miR390b was taken as the object. Based on the miR390b precursor sequence, the full length of the miR390b primary transcript was obtained by 5' RACE and 3' RACE. Both the full-length transcript and the corresponding DNA sequence of MIR390b were 584 bp, indicating that there is no intron in MIR390b of P. deltoides (Fig. S3, see online supplementary material). The transcription start site was located 89 bp upstream of the mature miR390b, and MIR390b was localized on chromosome 06 (Chr06) of the P. deltoides genome (Fig. 2B). Although the precursors of miR390b were less conserved among different poplar varieties, all precursors produced identical mature sequences (Fig. 2C). RNA from different tissues of Populus tremula × Populus alba INRA clone 717 1-B4 (hereinafter referred to as 717 hybrid poplar) was extracted and assayed for quality (Fig. S4, see online supplementary material). The expression level of miR390b was higher in primary root (PR) and petiole (PT) of 717 hybrid poplar, and lower in young stem (YS) and mature leaf (ML), especially in young leaf (YL) and AP (Fig. 2D).
To further understand the expression pattern of MIR390b, a βglucuronidase (GUS) construct driven by the promoter of MIR390b was transformed into wild-type (WT) 717 hybrid poplar. GUS staining was detected in AP and YL of 6-week to 8-week-old tissuecultured seedlings as well as in YS, and also in the main veins of ML, primordia of LR, and vascular tissue of LR and PR (Fig. 2E). Furthermore, weak GUS staining was detected in the primary root tip (PRT) and no staining in the lateral root tip (LRT) (Fig. 2E). In 6-week to 8-week-old soil-cultured plants, GUS staining was detected in epidermal trichome and epidermal cells of the third petiole (PT3) as well as in the primary xylem vessels (Fig. 2F). The expression of miR390b was also observed in the epidermal cells of PT9, but not in PT15 (Fig. 2F). From IN3 to IN9, the expression of miR390b in the epidermal cells of the stem gradually decreased and was eventually undetectable (Fig. 2F). With the transition of stem secondary growth, the expression of miR390b gradually increased in the primary xylem and even in the medullary rays of IN15 (Fig. 2F). The expression of miR390b in tissues continuously changed in a gradual manner with the growth of poplar, ref lecting its spatiotemporal specificity.

Overexpression of miR390b promotes stem elongation in poplar
To determine whether miR390 participates in the AP development of poplar, WT 717 hybrid poplar was transformed with the vector overexpressing (OE) miR390b for phenotype observation and identification (Fig. S5A, see online supplementary material). In the nine independent OE-miR390b lines obtained, the expression levels of miR390b were up-regulated in leaves ( Fig. S5B and C, see online supplementary material). Among them, three OE-miR390b , and LF samples. Tree (T) 1 to T3 represent the three biological replications. C Heat map of 17 differentially expressed miRNA during stem development and 23 highly expressed miRNA in LF. T1 to T3 in the sample names represent the three biological replications of the samples. Expression amount is plotted in scaled TPM using the pheatmap package in R. D Accumulation of the mature miR390a-c and miR390d in different samples by RT-qPCR. Data are expressed as mean ± SD (n = 3). Statistical significance was determined by one-way ANOVA. Significant differences between means (Duncan, P < 0.05) are indicated by lower-case letters above the bar. lines (i.e. OE-42, OE-44, and OE-65) with the significantly upregulated expression levels of the precursor and mature miR390b were selected for the subsequent experiments (Fig. 3A, Fig. S5D, see online supplementary material). After WT and OE-miR390b lines were moved into the greenhouse for growth, it was found that the OE-miR390b lines were higher than the WT plants (Fig. 3B). The growth data of plants were measured at a fixed time every week, and the results showed that the lengths from apex to node 10 of OE-miR390b lines were about 30% longer than that of WT (Fig. 3C). Although there was no consistent significant difference in the height growth and node number increment each month for OE-miR390b lines compared to that of WT ( Fig. S6A and B, see online supplementary material), the ratio of monthly growth of height to node number was significantly increased (Fig. 3D). In addition, the significant increase in the ratio of plant height to node number in the OE-miR390b lines was caused by a significant increase in plant height rather than an increase in the total node number of the whole plants ( Fig. S6C-E, see online supplementary material). To be specific, each IN length of OE-miR390b lines from IN5 to IN10 was significantly longer than that of WT ( Fig. 3E and F), without significant differences in base diameter and IN diameter ( Fig. S6F and G, see online supplementary material). There were no significant differences in leaf morphology, leaf area, and leaf aspect ratio between the OE-miR390b lines and WT ( Fig. S7A-C, see online supplementary material). In addition, no significant differences were observed in root morphology, root dry weight, and root fresh weight  The height of the capital letter at each position in the sequence logo represents the degree of conservation. B Localization of MIR390b gene in the poplar genome. The blue box indicates the full-length of MIR390b gene, the orange box indicates miR390b precursor and the red box indicates mature miR390b. C Sequence alignment of miR390b precursor in different poplar species. The red box indicates the sequence of mature miR390b. D The expression level of miR390b in different organs of WT by RT-qPCR. AP, apex; LR, lateral root; ML, mature leaf; OS, old stem; PR, primary root; PT, petiole; YL, young leaf; YS, young stem. Data are presented as means ± SD (n = 3). Statistical significance was determined by one-way ANOVA. Significant differences between means (Duncan, P < 0.05) are indicated by lower-case letters above the bar. E and F Histochemical staining of transgenic poplars harboring the GUS reporter gene driven by the promoter of MIR390b. The 6-week to 8-week-old tissue-cultured seedlings (E) and 6-week to 8-week-old soil-cultured seedlings (F) were used for GUS staining. LRT, lateral root tip; PRT, primary root tip. The scale bars are 500 μm in E and 100 μm in F.
stem. Based on said factors, the development of vascular tissue in soil-cultured plants were observed in transverse sections, and the lengths of xylem fiber cells (which had undergone cell wall thickening) were measured in longitudinal sections ( Fig. 3G and H). The vascular development state of IN4 in OE-miR390b lines was essentially similar to that of IN6 in WT, and secondary growth of xylem fiber cells had occurred in IN7 of all OE-miR390b lines and WT (Fig. 3G). The measurement and statistical analysis of the xylem fiber cell lengths of IN8 revealed that the lengths of xylem fiber cells in OE-miR390b lines were 7-10% shorter compared with that of WT ( Fig. 3H and I). However, the relative numbers of IN8 xylem fiber cells in the longitudinal axis were 37-47% higher in OE-miR390b lines than that in WT (Fig. 3J). Such results demonstrate that overexpression of miR390 promoted the stem elongation and plant height and increased the number of xylem fiber cells in the longitudinal axis.

Genome-wide identification of genes in response to miR390 overexpression in apex tissue
Plant SAM determines the number of cells in the longitudinal direction of stem internodes through the regular production of lateral organs, which subsequently affects the length of INs. To explore the downstream pathway of miR390-mediated stem elongation, transcriptome data from the AP tissues of three OE-miR390b lines and WT were compared. On average, over 75% of clean reads of each sample were specifically aligned to the reference genome of P. trichocarpa (Table S3, Table S4, see online supplementary material). The heat map shows that four DEGs were up-regulated and 23 DEGs were down-regulated in three OE-miR390b lines (Fig. 4C). Such results indicate that the overexpression of miR390b in poplar affects the expression levels of related genes and mainly plays a negative regulatory role.

TAS3.1 and TAS3.2 are the target genes of miR390 in poplar
As an endogenous non-coding small RNA, miRNA usually regulates plant growth and development by cleaving mRNA of downstream target genes. Thus, identification of miR390 target genes is of great significance for analysing its function in regulating stem elongation. Four TAS3 genes, namely TAS3.1 (Potri.010G149600), TAS3.2 (Potri.008G101675), TAS3.3 (located on Chr18), and TAS3.4 (Potri.002G191950), were identified as candidate targets of miR390 in poplar [34]. Among said genes, TAS3.2 belonged to one of the DEGs shared by the three OE-miR390b lines (Fig. 4C). The candidate target genes TAS3.1 and TAS3.2 were highly homologous and had highly similar sequence structures (Fig. 5A). Consistent with previous reports [34], there was a G:U wobble base pair of the 11th nucleotide and a mismatch of the 12th nucleotide at the 5 target site (TS), as well as a mismatch of four nucleotides at the 3 TS in poplar (Fig. 5A). Based on the complementarity of the two TSs with miR390, and combined with previous reports [19], an assumption was made that the TS of miR390 in poplar may be conservatively located at the 3 proximal end of the candidate targets TAS3.
To verify whether miR390 could directly cleave the 3 proximal end TSs of the two potential target genes, transient coexpression in Nicotiana benthamiana leaves was used (Fig. 5B-J). Compared with three negative control groups, the green f luorescent protein (GFP) f luorescence intensity of co-injection with OE-miR390b + TAS3.1/TAS3.2-3 TS-GFP (i.e. b + d/e) was significantly reduced (Fig. 5C, E, G, and I). However, the GFP f luorescence intensity of co-injection with empty vector + TAS3.1/TAS3.2-3 TS-GFP (i.e. a + d/e) was also obviously less than the two other negative controls. In tobacco, the miR390 family has three members (miR390a-c), of which the mature sequences of miR390b and miR390c are identical to that of poplar miR390a-c and miR390d-5p, and the mature miR390a differs from the poplar miR390 by only one base difference at the 3 proximal end (Fig. S9, see online supplementary material). Therefore, an assumption was made that tobacco miR390 may cleave vectors carrying the 3 proximal end TS of potential target genes. As such, the GFP f luorescence intensity of tobacco leaves co-injection with STTM-miR390 + TAS3.1/TAS3.2-3 TS-GFP (i.e. c + d/e) was significantly higher than that of OE-miR390b + TAS3.1/TAS3.2-3 TS-GFP and empty vector + TAS3.1/TAS3.2-3 TS-GFP (Fig. 5D, F, H, and J). The results provide evidence that TAS3.1 and TAS3.2 are the target genes of miR390 in poplar.
In WT, the expression level of TAS3.1 was significantly lower in PR and AP, but higher in YS and ML (Fig. 6A). Except in YS and AP, the expression level of another target gene TAS3.2 was low in other poplar tissues (Fig. 6B). The expression of miR390b was significantly up-regulated in different tissues of OE-miR390b lines, especially in ML, YL, and AP, which was over 100-fold higher than that of WT (Fig. 6C). Target genes TAS3.1 and TAS3.2 exhibited different levels of down-regulated expression in OE-miR390b lines ( Fig. 6D and E). TAS3.1 was significantly down-regulated in LR, YS, and YL of three OE-miR390b lines and down-regulated in PR, old stem (OS), ML, and AP of individual OE-miR390b lines . Asterisks indicate statistically significant differences using Student's t-test ( * * P < 0.01).
( Fig. 6D). Except for the expression in PR of OE-42 and OE-65, which did not exhibit consistently significant differences, TAS3.2 was significantly down-regulated in all other tissues of three OE-miR390b lines and was more down-regulated in AP (Fig. 6E). Such results indicate that the overexpression of miR390b effectively suppressed the expression levels of target genes TAS3.1 and TAS3.2 in poplar, and the suppression effect on TAS3.2 was more significant in YS and AP.

Prediction of ARFs regulated by miR390-TAS3 in Populus
To predict which ARFs were downstream-regulated by miR390-TAS3 during stem elongation in Populus, phylogenetic analysis based on ARF proteins from Populus and Arabidopsis were conducted, gene expression of the predicted ARFs was analysed by RT-qPCR, and correlation analysis between OE-miR390b phenotypes and whole-genome gene expression levels was performed by weighted correlation network analysis (WGCNA). In P. trichocarpa genome, a total of 36 genes are annotated as ARF proteins. The full-length of such protein sequences together with ARF2, ARF3, and ARF4 protein sequences from Arabidopsis were used to construct a phylogenetic tree (Fig. S10, see online supplementary material). Proteins of Potri.012G106100 and Potri.015G105300 belonged to the same branch as Arabidopsis ARF2, indicating the highest similarity, referred to as ARF2.1 and ARF2.2, respectively. Meanwhile, proteins of Potri.004G050150 and Potri.011G059300 belonged to the same branch as Arabidopsis ARF3, and Potri.009G011800 and Arabidopsis ARF4 belonged to the same branch, referred to as ARF3.1, ARF3.2, and ARF4, respectively. Although Potri.011G059101 was also predicted to be an ARF3/4 [34], it was omitted in the next study because of the distant evolutionary relationship with Arabidopsis ARF3/4. The expression levels of five ARFs in poplars were detected by RT-qPCR under the effect of overexpression of miR390b. The results showed that the transcription levels of ARF2.2, ARF3.2, and ARF4 were significantly repressed in three OE-miR390b lines (Fig. 6G, I, and J). Although the expression levels of ARF2.1 and ARF3.1 were significantly repressed only in individual OE-miR390b lines, the expression levels in other lines also exhibited a down-regulation trend ( Fig. 6F and H). The expression trends of ARF genes in RNA-Seq data were basically consistent with the results of RT-qPCR (Fig. 6F-J). The co-expression relationship between the gene expression levels from RNA-Seq and multiple phenotypic data of OE-miR390b lines was speculated through the correlation coefficient, and the downstream key ARFs of miR390 promoting stem elongation were screened. Given the negative regulatory effect of miR390 on ARFs, ARFs that were negatively correlated with IN length and the number of fiber cells, and positively correlated with the length of fiber cells were focused on. The five ARF genes of poplar were distributed in three modules, ARF3.2 and ARF4 in the pink module were most strongly correlated with the above three phenotypes (Fig. 6K;  Fig. S11, see online supplementary material). The results reveal that ARF3.2 and ARF4 responded to the overexpression of miR390b and cooperated with miR390 to participate in the stem elongation in poplar.

Identification of microRNAs involved in poplar stem development
sRNA-Seq was performed on APs and INs from 9-year-old P. deltoides, with LF samples serving as controls. A total of 17 known miRNAs were differentially expressed during stem development (Fig. 1C), suggesting their potential roles in AP and vascular tissue development. Specifically, miR171 was expressed and enriched in AP, which is consistent with the previous report on Arabidopsis [35]. The miR171 function of regulating SAM development by repressing target genes HAIRY MERISTEMs (HAMs) has been demonstrated in several species [36][37][38]. In the present study, miR164 was enriched in IN9 of Populus. A previous study reported that overexpression of its target gene NAC (NAM, ATAF, and CUC) in Arabidopsis delayed stem elongation [39]. miR396, also enriched in IN9, has been reported to be involved in regulating internode elongation through the mediation of the target genes GRF in several species [40][41][42]. Furthermore, miR172, with relatively high expression in IN3 and IN4, has been reported to regulate internode elongation during the reproductive stage in rice [43]. Therefore, an assumption was made that differentially expressed miRNAs in AP or stem may be involved in the regulation of growth and stem development in Populus. The mature sequence of miR390a-c and miR390d-5p were specifically abundant in AP and IN9. The results of GUS histochemical analysis further reveal the high activity of the miR390b promoter in AP (Fig. 2E). Regulatory pathways consisting of miR390 and its downstream target genes TAS3 and ARFs have been reported in various aspects of plant growth and development, as well as in response to biotic and abiotic stresses [21][22][23][24][25][26][27][28][29][30][31][32]. Although miR390-TAS3-ARFs module has been reported to be involved in the regulation of lateral root growth [33], so far, miR390 function has been less studied in poplar. Poplar is one of the main sources of timber and has an important economic value. However, the functional research of miR390 in poplar stem development remains relatively limited. A series of findings in this study showed that overexpression of miR390b affected the plant height and internode length in poplar. We discovered a novel function of miR390b in poplar involved in regulating stem elongation and affecting woody biomass production, which has important implications for the field of tree biotechnology.

The regulation of stem length by miR390-TAS3 in poplar is related to the activity of shoot apical meristem
In the OE-miR390b lines, the trend in the expression level of mature miR390b was consistent with the trend in the number of DEGs (Figs 3A and 4B). It is worth noting that the three OE-miR390b lines showed low overlap of DEGs. There could be several reasons. The three lines are derived from three independent transgenic events. Changing the expression level of mature miR390b in the three lines result in less than 2-fold of change in the expression of the target ARFs, which might lead to subtle differences in their downstream network in regulation of organogenesis. In addition, collecting the whole apex as the sample for RNA-Seq might exclude some potential DEGs with high cell type-specific expression. As one of the overlapping 27 DEGs, TAS3.2 was significantly down-regulated in the majority of tissues of three OE-miR390b lines, suggesting that TAS3.2 may be a major target gene for stem elongation in Populus. Combined with the expression levels of the downstream ARF3.2 and ARF4 and correlation analysis with the phenotype of OE-miR390b lines, we come to the hypothesis that the conserved miR390b-TAS3-ARFs regulatory pathway may mediate stem elongation in poplar.
It is well known that the increase of bioactive GA level can promote the elongation and growth of plant stems. In Populus, several reports have verified that overexpression of GA20 oxidase, a key enzyme for producing bioactive GA, can significantly increase plant height and stem length [4,[44][45][46]. However, in our study, the increase in stem internode length was not positively correlated to the length of xylem fiber cells (Fig. 3), which is not the same scenario as the previous reports [4,5,45]. Furthermore, the pleiotropic effects of elevated GA leading to smaller leaf area and lower root weight in poplar [44][45][46] was also not observed in the OE-miR390b lines (Fig. S7, see online supplementary material). Therefore, although the OE-miR390b lines resulted in internode elongation, GA signaling pathway may not be directly correlated.
Plant SAM continuously generates new organs in an orderly manner, and the daughter stem cells generated by directed cell division are the source of the vascular cambium. The speed at which SAM produces lateral organs directly determines the number of procambial cells in the stem, which in turn affects the number of vascular cells along the longitudinal axis of the stem internode. It is hypothesized that miR390b-TAS3-ARFs regulatory pathway affects the stem elongation growth of poplar may be related to SAM development. In a recent study, ARF3 and ARF4, mediated by the histone deacetylase HDA19, repressed SHOOT-MERISTEMLESS (STM) expression and promoted the initiation of reproductive primordia [47]. Down-regulation of STM is also an early marker for the formation of leaf primordia in SAM [48]. Moreover, STM is specifically expressed in SAM and is a significant regulatory factor in the maintenance of meristem stem cells [49]. In the present study, the down-regulated expression of ARF3.2 and ARF4 in AP may affect the expression of STM and thereby disturb the homeostasis of cell division and cell differentiation in AP. The disruption may also have led to an increase in the cell number between leaf primordia in the longitudinal direction, or an increase in the cell number of the procambium and/or the precursor cells thereof (Fig. 6L). STM was not identified as a differentially expressed gene in the present RNA-Seq analysis, which could be due to its fine-tune regulation or cell-type specificity. The development of specific cellular markers for poplar AP will facilitate precise identification of the cell types involved in miR390b promoting cell division in AP. Additionally, the rapid development of single-cell RNA-Seq technology will also provide new solutions for cell type-specific gene expression analysis.

Functional diversification of miR390-TAS3 in Populus
Multiple studies have shown that miR390-TAS3 is involved in regulating LR growth in a variety of plants [22,23,29], including Populus [33]. However, in the present study, no significant difference was observed in LR development in OE-miR390b plants compared with WT. Similarly, overexpression of Osta-siR2141 and OsTAS3a both resulted in the down-regulation of OsARF3 in rice. Compared with WT, root morphology was not significantly different in plants overexpressing Osta-siR2411, whereas overexpression of OsTAS3a significantly increased LR formation [30,50]. Therefore, an assumption was made that miR390-TAS3 may be involved in regulating different aspects of plant growth and development in different species and even different varieties.
ARF3 and ARF4 were the positive regulators that maintain cambial activity [51]. In the present study, the expression levels of TAS3.1 and TAS3.2 were higher in YS of 717 hybrid poplar, and both of them were significantly inhibited by miR390b in the OE-miR390b lines. However, no significant changes in the cell morphology of the vascular cambium were observed in said lines. The inhibitory effect of miR390b-TAS3 on ARF3.2/4 in AP was significant but relatively mild. The cell-type specificity of TAS3.1 and TAS3.2 expression in the stems and the intercellular transport of miR390b and tasiARFs remain unclear. Even though miR390-TAS3 can inhibit the expression of ARF3 and ARF4 in the cambium, whether its activity is sufficiently suppressed in the cambium needs to be further investigated.
In summary, overexpression of miR390b can promote stem elongation and height growth in 717 hybrid poplar. Our results suggest that the miR390b-TAS3.1/TAS3.2-ARF3.2/ARF4 pathway is involved in regulating these biological processes. The involvement of miR390b-TAS3 in regulating tree height growth adds a new function to the miR390 family in regulating plant growth and development, which is also of great significance for tree biotechnology research.

Plant material and growth conditions
For sRNA-Seq, 9-year-old P. deltoides clones were grown at a field site in Huazhong Agricultural University (Wuhan, China).
The tissue-cultured seedlings of 717 hybrid poplar and transgenic lines were grown on 1/2MS medium, subcultured by microcutting regularly every two months in a tissue culture room with a photoperiod of 16 h light and 8 h dark at 25 • C. Two-monthold, robust tissue-cultured seedlings of WT and transgenic plants were transferred to the poplar growth room for 8 weeks with soil culture, then transplanted to 16 × 18 cm large pots and grown in a greenhouse under natural light conditions from 10 • C to 35 • C. N. benthamiana plants were grown in the plant growth room with long-day conditions (16 h light and 8 h dark) at 23 • C.

sRNA-Seq analysis
For sRNA-Seq, samples of the AP (contains SAM), stem segments of IN2-IN5 (no obvious secondary growth occurred in the vascular tissues), IN9 (contains mature vascular tissue), and LF without midrib were collected from one branch ( Fig. 1A; Fig. S1, see online supplementary material). Three branches from three independent 9-year-old P. deltoides trees (T1, T2, and T3) were selected as three biological replicates, respectively. IN samples were collected in lengths of 2 mm and sample AP was collected from apical buds (Fig. 1A). Total RNA was separately isolated using Trizol reagent (Invitrogen, China). Libraries for sRNA-Seq were generated as described in the NEBNext Multiplex Small RNA Library Prep Set for Illumina after total RNA quality control. The libraries were sequenced on the Illumina HiSeq 2500/2000 platform, and single-end reads with 50 bp were generated.
Clean reads were obtained from raw data after removing lowquality reads, reads with 5 adapter contaminants, and reads without 3 adapter or the insert tag. The certain range (18-30 nt) of lengths from the clean reads were mapped to the reference P. trichocarpa v3.1 genome by Bowtie without mismatch. Known miR-NAs were identified by aligning clean reads with the miRBase22.1 database (https://mirbase.org/). Unknown sRNA were obtained by excluding (i) non-coding RNA sequences (rRNAs, tRNAs, snRNAs, and snoRNAs) using NCBI (http://www.ncbi.nlm.nih.gov/) and Rfam (http://www.sanger.ac.uk/resources/databases/rfam.html); (ii) repeat sequences using a repeat sequence database (http:// www.repeatmasker.org/cgi-bin/WEBRepeatMasker/webcite); and (iii) tags originating from protein-coding genes by mapping to the exon and intron of mRNAs of Populus. The potentially novel miRNAs were predicted with miRDeep-P from the remaining unknown sRNA. The abundance of miRNA was generated based on transcripts per million (TPM) values. Differential expression analysis of miRNAs among the samples was performed using Rpackage DEseq2, with the threshold for significant differential expression of absolute value log2 (fold change) ≥1.0 and adjusted P value <0.05. Expression amount is plotted in scaled TPM using the pheatmap package in R. Scaled TPM values were obtained by scaling TPM of each miRNA among samples within each biological replicate, and 0.01 was added to the TPM values for LF samples before scaled.

Gene cloning and sequence analysis
Total RNA was extracted from the IN9 of 9-year-old P. deltoides using the improved 2× CTAB method and reverse transcribed into cDNA in accordance with the manufacturer's instructions. According to the precursor sequence of miR390b, the full-length transcript of MIR390b was obtained from IN9 cDNA of P. deltoides by SMARTer ® RACE 5 /3 Kit Components (TaKaRa, China) using the primers listed in Table S1 (see online supplementary material). Subsequently, the full-length sequence of miR390b was amplified from IN9 gDNA of P. deltoides using primers miR390b-Full-F/R (Table S1, see online supplementary material).
The sequence conservation of all the members of the miR390 family in land plants was analysed by the online software WEBLOG. Software ClustalX and GeneDoc were used for sequence alignment and editing alignment results, respectively. The precursor sequences of miR390b in Populus euphratica, Populus tomentosa, and P. trichocarpa were downloaded from the online database NCBI (https://www.ncbi.nlm.nih.gov/) and in 717 hybrid poplar from the online database AspenDB (http://aspendb.uga. edu/index.php/databases/spta-717-genome). The full-length sequences of TAS3 genes and ARF proteins were obtained from the P. trichocarpa v3.1 genome (https://phytozome-next.jgi.doe. gov/). The phylogenetic tree for the full-length sequence of ARF proteins was constructed using MEGA5.

Construction of expression vector and generation of transgenic lines
For the construction of the GUS fusion vector, a 2587 bp promoter fragment upstream of the miR390 coding gene start site was amplified from P. trichocarpa gDNA, cloned into the entry vector pDONR201 (Invitrogen, China), and then recombined into the upstream of GUS protein of the expression vector pKGWFS7. For the construction of the overexpression vector, the full length of MIR390b was ligated to the entry vector pGWC and then recombined into the overexpression vector pH2GW7, a plasmid carrying the 35S promoter and a hygromycin-resistance gene, using the Gateway LR reaction (Invitrogen, China).
The expression vectors were transformed into Agrobacterium tumefaciens and then stably transformed into 717 hybrid poplar by leaf disc transformation [52]. The sequence fragments of reporter genes or/and target genes in the expression vector were amplified by PCR to screen and identify the transgenic positive lines (Table S1, see online supplementary material).

Gene expression assays
For the quantification of the miR390b precursor, total RNA was extracted from the fourth leaf at the top of the 4-weekold tissue-cultured seedlings of transgenic plants using the Ultrapure RNA Kit (CWBIO, China). For tissue-specific expression quantification of related genes, LR, PR, OS, YS, PT, ML, YL, and AP of 13-week-old soil-cultured plants of transgenic plants were collected. Based on the characteristics of tissue samples, total RNA was extracted from OS and PT using TransZol (TRANS, China), from YS, YL, ML, and AP using the Ultrapure RNA Kit, from PR and LR using the HiPure Plant RNA Mini Kit (Magen, China) according to the manufacturer's protocols. RNA quality was evaluated using the NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, Delaware, USA) and semi-quantitative RT-PCR.
The transcription levels of genes were detected using RT-qPCR. RNA samples were treated with the gDNA eraser and then reverse-transcribed into cDNA using the primeScript RT reagent Kit (TaKaRa, China) according to the manufacturer's instructions, with PtActin as the reference gene. The RT-qPCR was performed using 2× ChamQ Universal SYBR qPCR Master Mix (Vazyme, China) and gene-specific primer pairs (Table S1, see online supplementary material) on a Roche LightCycler 480 II, according to the manufacturer's instructions. The assays were performed using three independent biological replicates, each comprising three technical replicates.

GUS staining
Different tissues of transgenic plants were added with pre-cooled 90% acetone (v/v) and fixed at 4 • C for 20 min. The samples were washed twice with potassium phosphate buffer for removing acetone, immersed in X-Gluc solution for treatment at 37 • C in the dark for 5-24 hours, and washed with 70% alcohol every 3-4 hours until the WT tissues were completely decolorized. The tissue samples of the cultured seedlings were observed under a stereoscopic microscope (Leica, Germany) and photographed for preservation.

Length measurement and number estimation of fiber cell
The stem internodes were transversely and longitudinally sectioned using an automatic vibration slicer (Leica, Germany). The sections were stained with 0.05% (w/v) aqueous toluidine blue reagent for lignin characterization, then observed and photographed under a f luorescence microscope (Olympus, Japan).
The length of xylem fiber cells was measured by ImageJ software. A total of 120 fiber cells of each IN8 were selected for measurement, and each line had three independent plants acting as three biological replicates. The number of fiber cells in each IN8 was estimated by the ratio of IN8 length to the average length of fiber cells [53].

RNA-Seq analysis
To compare gene expression profiles, AP tissue samples (about 5 mm long) of 13-week-old soil-cultured plants of WT and three OE-miR390b lines were collected. Removal of gDNA from total RNA using Recombinant DNase I (RNase-free) (TaKaRa, Japan). The RNA-seq libraries were constructed using NEBNext ® UltraTM RNA Library Prep Kit for Illumina ® (NEB, Santiago, CA, USA). The Agilent 2100 bioanalyser was used to detect the size of the library insert and the effective concentration of the library was accurately quantified by RT-qPCR The prepared DNA Nano Ball was loaded onto the sequencing chip and the transcriptome was sequenced in Illumina Hiseq PE150 (Frasergen, China). Raw reads were filtered to obtain high-quality clean reads. The quality of clean reads was detected using FastQC software and high-quality reads were mapped to the reference P. trichocarpa v3.1 genome using HISAT2 software. The raw read counts of each gene were calculated by HTSeq, followed by identification of DEGs using Rpackage DEseq2, with the criteria of the absolute value log2 (fold change) ≥1.0 and adjusted P-value <0.05.

Transient co-expression assay
The transient co-expression assay was performed as described previously [54]. The short tandem target mimic sequence of miR390 (STTM-miR390) was synthesized [55]. The TSs of miR390 candidate target genes were cloned into the pMS4v2 vector (between XhoI and XbaI sites) carrying the GFP coding gene and driving expression using the caulif lower mosaic virus 35S promoter. A mutant TS (tas3) that could not be cleaved by miR390 was also cloned into the pMS4v2 for negative control.
The equivalent of the two A. tumefaciens (GV3101) suspensions were mixed and co-injected into fully expanded tender leaves of 5-week-old N. benthamiana. The GFP signals were observed using a handheld UV analyser 48-72 h after infiltration. The sequencespecific primers for constructing said plasmids in the present experiment are shown in Table S1 (see online supplementary material).

Statistical analysis
In all experiments of the present study, at least three independent plants with the same age and cultured conditions were used. Excel software (Microsoft) was used for the statistical analysis of the data with at least three biological replicates and three technical replicates. Statistically significant differences were determined by two-tailed and twosample Student's t-test ( * P < 0.05, * * P < 0.01), or assessed by analysis of ANOVA followed by Duncan's multiple comparisons (P < 0.05).

Data availability
The sRNA-Seq data and RNA-Seq data have been deposited in the NCBI Sequence Read Archive under BioProject accession numbers PRJNA833047 and PRJNA833269, respectively.